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doi: 10.1242/10.1242/dev.00116

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The Drosophila cytokine receptor Domeless controls border cell migration and epithelial polarization during oogenesis

Christian Ghiglione^*, Olivier Devergne^*, Emmanuelle Georgenthum, Fabrice Carballès, Caroline Médioni, Delphine Cerezo and Stéphane Noselli

Institute of Signaling, Developmental Biology and Cancer, UMR 6543 — CNRS, Parc Valrose, 06108 NICE cedex 2, France

View larger version (80K): [in a new window]   Fig. 1. Domeless controls border cell migration and interacts with the JAK/STAT pathway. (A) Schematic representation of BCs (in red) during stages 8 to 10. The BC cluster, containing the polar cells (in black), starts to delaminate from the follicle cell layer (in grey) during stage 9 and invades the nurse cell compartment (in blue). At stage 10, BCs reach the anterior of the developing oocyte (in yellow). (B) Wild-type stage 10 egg chamber stained with phalloidin-FITC (green; staining the actin cytoskeleton) and propidium iodide (red; staining the nuclei). Note the round BC cluster at the anterior of the oocyte (arrowhead). (C) Stage 9 egg chamber from an heterozygous domePL100/+ female. The BC cluster is not correctly assembled with some cells being left behind (arrowheads). (D,E) Mosaic egg chambers containing dome mutant clones (recognized by the absence of the GFP clonal marker and upregulation of the Fas3 marker (in red; see also Fig. 7). Loss of dome in BC precursors leads to defective, or absence of, BC migration. For comparison, the normal position of the cluster as it would be in a wild-type chamber is represented by a dashed circle. (F,G) Early stage 2-3 dome egg chambers showing incomplete encapsulation (arrowheads, F) or fusion (G). Anti-Dome staining is in red. (H) dome interacts genetically with Stat92E and dpias. Histogram representing the percentage of egg chambers of various genotypes with BC migration phenotypes similar to those shown in C or with defective BCs at stages 9 and 10. (I,J) dome homozygous mutant follicle cells (GFP negative) show a dramatic reduction in nuclear Stat92E (in red). Anterior is to the left.

View larger version (73K): [in a new window]   Fig. 2. Expression of upd and dome in egg chambers. In situ hybridization using upd (A,B) or dome (C,D) antisense riboprobes. The upd mRNA is detected in the 2 pairs of polar cells and is preferentially accumulated in the apical region (B, arrowheads). The dome transcripts are widely and weakly expressed in follicle and germline cells (C). (D) Overexpression of dome using the slbo-GAL4 line and a UAS-dome construct.

View larger version (144K): [in a new window]   Fig. 3. Dome subcellular localization in follicle cells. (A,B) Immunostaining of dome mosaic egg chambers using an anti-Dome antibody (in red). Mutant clones are identified by the loss of the GFP clonal marker (dashed lines; see Materials and Methods). The Dome protein is targeted to the apicolateral membranes in all follicle (A,B) and germline cells (not shown). Membrane staining is absent in cells that are mutant for 2 different dome alleles (domePG14, dome12030). A dome-GFP fusion protein is also targeted to the membrane (C; red is phalloidin-TRITC). Overexpression of Dome-GFP (C), wild-type Dome (D) or a truncated Dome (domeEXT; E), promotes the formation of Dome-containing intracellular vesicles apically.

View larger version (98K): [in a new window]   Fig. 5. Structure-function analysis of the Dome receptor. The migratory phenotype of BCs after overexpression of the JAK/STAT pathway components is analysed using slbo-lacZ as a reporter, which is expressed primarily in the BCs (arrowhead) at stages 9 and 10. (A) At stage 10, slbo-lacZ is also expressed in centripetal and posterior polar cells. (B-L) Overexpression, using a slbo-GAL4 line (Rorth, 1998), of Hop (B), Stat92E (C) or Upd (M) leads to aberrant BC migration. To test the domain requirements of Dome, several different constructs have been expressed (D) and the resulting phenotypes analysed (E-L, arrowheads indicate BCs). Overexpression of a wild-type Dome or of a Dome-GFP fusion protein leads to an absence of migration of BCs (E-H). Expression of truncated forms (DomeCYT or DomeEXT) blocks the recruitment of BCs, and only polar cells express slbo-lacZ (I-L). Note that even in late stage egg chambers (L; stage 11) polar cells are still present at the anterior pole. Co-expression of Upd and the dominant negative DomeCYT proteins in BCs completely suppresses the gain-of-function phenotypes associated with Upd overexpression (compare M and N). The frequency of BC migratory defects is indicated in the lower right corner of each panel. Anterior is to the left.

View larger version (98K): [in a new window]   Fig. 4. Upd-dependent internalization of Dome. From stages 4 to 10, endogenous Dome (in red) is detected in intracellular vesicles. (A,B,B') In early stages, vesicles are present in all follicle cells (A is a stack of several optical sections; B,B' represent an optical section through a stage 4-5 egg chamber), but later, they become restricted to the poles of the egg chambers (C,F-I), where the Upd ligand is most concentrated. (C-E) BCs contain a high level of Dome vesicles before, during, and after migration. (G,I,F,H) Dome is expressed in a gradient (represented by the white wedges) of apically located vesicles at the anterior (G,I) and posterior poles (F,H). (B,H,I) Vesicles contain the Dome protein, since they are absent in dome mutant follicle cells (broken outlines) at any stage. (J-L) Ectopic expression of the Upd ligand in main body follicle cells (J,K) or BCs (L) causes the accumulation of extra Dome-containing vesicles.

View larger version (104K): [in a new window]   Fig. 6. Regulation of dome expression by the JAK/STAT pathway. In hop mutant follicle cells, Dome protein level is up-regulated (A; Dome is in red). dome-GAL4 is a pGAL4 element inserted upstream to the dome transcription start site (E) that drives expression in BCs and posterior follicle cells (B). Overexpression of wild-type dome (C) strongly reduces the level of GFP driven by dome-GAL4 (compare B and C), while expression of domeEXT has no effect (D). In D, the reduction in GFP expression is due to the absence of BCs induced by the expression of domeEXT (see Fig. 5I). (E) Schematic view of the dome promoter showing the localization and sequence of 2 putative Stat92E binding sites.

View larger version (87K): [in a new window]   Fig. 7. Dome controls the expression of several follicle cell markers. Immunostaining (in red) of dome mosaic egg chambers using anti-DE-cadherin (A,B), anti-Fas3 (C-F) and anti-Crumbs (G-J) antibodies. (A,B) Confocal sections of two different egg chambers showing reduction of DE-cadherin. Fas3 is normally expressed in polar cells in wild-type egg chambers (C), and becomes overexpressed in dome mutant cells close to the anterior (E) or posterior (F) polar cells. dome mutant clones generated away from the poles also accumulate Fas3 (D). Mutant clones have been outlined (dotted lines in A,B,D,E,F and solid bar in G-J). Anterior is to the left.